The invention relates to a process for producing monoclonal antibodies to epizootic hemorrhagic disease virus antigen and to the hybrid cell lines that secrete these monospecific antibodies.
Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious, insect-transmitted virus disease that affects primarily wild ruminants and cattle. Epizootic hemorrhagic disease virus (EHDV) is classified as an Orbivirus and is antigenically related to bluetongue virus (BTV), also an Orbivirus. Two distinct serotypes of EHDV are known to exist in the United States, and seven or more exist worldwide. The two U.S. serotypes were initially isolated from clinically sick deer but are commonly isolated from cattle, wild ruminants, and the suspected vector, Culicoides variipennis.
EHDV infection of white-tailed deer and antelope is most often a peracute infection with a high rate of morbidity and mortality. The disease in cattle is usually subclinical with only mild signs similar to infection with BTV. However, economic losses due to EHDV infections in cattle result from restrictions to export markets and to a lesser extent, from clinical infection. Because EHDV and BTV clinical infections in cattle are so similar, a definitive diagnosis cannot be made without conducting proper laboratory tests.
Additionally, because these two viruses are antigenically related, that is, the antibodies produced by the animal in response to infection with one virus may cross-react in the laboratory when tested against the other one, to accurately diagnose EHDV or BTV it is necessary to isolate the viral agent and correctly identify the isolated virus.
One laboratory test that has been used to differentiate between EHDV and other viruses is the indirect fluorescent antibody test (IFAT), which uses hyperimmune rabbit serum taken from EHDV-inoculated rabbits to visualize viral antigen in virus-infected cell cultures. Because EHDV and BTV "share" antigenic sites on some of the viral proteins, even the most specific rabbit antibody to EHDV will contain antibodies that cross-react with BTV antigens. Recently, M. M. Jochim and S. C. Jones developed monospecific (monoclonal) antibodies which identify BTV in infected cell cultures and which do not react with EHDV antigen in infected cells. These are described in U.S. patent application Ser. No. 570,155. What is needed is a monoclonal antibody that will identify either of the two serotypes of EHDV in the same way that monoclonal antibodies have been produced and used to identify BTV. In this way the diagnosis of BTV-EHDV would be simple and complete with essentially no possibility of misidentification of these two closely related animal viruses.
The production of monoclonal antibodies by the fusion of spleen cells from immunized mice and myeloma cells grown in continuous culture, has been described previously, e.g., Kohler et al. in Nature, Vol. 256, pp. 495-497 (1975), Kohler et al. in European Journal of Immunology, Vol. 6, pp. 511-519 (1976), Galfre et al. in Nature, Vol. 266, pp. 550-552 (1977), and in the text Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, R. Kennett, T. McKearn, K. Bechtol, Eds., Plenum Press, New York and London (1980). Techniques for the chemical selection of hybridomas arising from such a fusion, and subsequent isolation of single antibody secreting cell clones for the production of the monoclonal antibodies are also known. However, no cell lines have been produced capable of secreting monoclonal antibodies which are group-specific to EHDV and not serotype-specific, that is, which recognize either EHDV serotype 1 or serotype 2, which do not give false positive reactions to closely related viruses such as BTV, and which have been shown to be useful for identification of EHD viral antigens using common, simple tests such as immunofluorescence.
It should be noted that because of the unpredictable nature of hybrid cell preparation, one cannot extrapolate from one antigen or cell system to another.